Recently, factor VIII preparations which have been purified from large plasma pools by means of ion exchange chromatography, or very recently by means of immunoaffinity, have been made available to haemophilics in adequate quantities.
Various preparations of FVIII which have been obtained by genetic manipulation are currently under development or under clinical trial. These FVIIIs are either intact molecules or deleted molecules (Bihoreau (1992)).
FVIII is a glycoprotein cofactor of plasma coagulation and acts at the level of factor X (FX) activation. Characterization of FVIII and its mechanism of action is made more difficult because of its low concentration in the plasma, the size heterogeneity and its extreme sensitivity to enzymic degradation. This reaction comprises the proteolysis of FX to form activated factor X (FXa=Stuart factor) and brings into play a complex (Tenase complex) which comprises the enzyme (activated FIX or FIXa), a cofactor (activated FVIII or FVIIIa), calcium ions and phospholipids.
FVIII is a protein which is so complex that, even though the sequence of its gene has been known since 1984 (Vehar et al. 1984 Nature 312, pp. 337-342), neither the complete structure of plasma FVIII (only about 50% of the protein has been sequenced) nor the precise structure of the carbohydrates has yet been established. The DNA sequence has been allowed to define the primary sequence of FVIII (SEQ ID NO: 21) (a rare exception to the instructions laid down by the FDA for the therapeutic products derived from biotechnology).
Nevertheless, subtle differences between plasma FVIII and recombinant FVIII have been identified: i.e. glycosylation, plasma half-life following infusion, etc.
FVIII is in the main synthesized in the hepatocytes. It has been cloned in mammalian cells, insect cells and yeast cells (Webb et al., 1993). These glycoproteins which are produced by biotechnological processes can exhibit differences in the structure and composition of the sugars as compared with the natural protein. The cDNA of FVIII has also been expressed in transgenic sheep (Halter et al., 1993).
The cDNA encodes a polypeptide of 2351 amino acids, including the signal peptide of 19 amino acids which is cleaved off in the endoplasmic reticulum. Post-translational modifications take place in the Golgi apparatus: i.e. glycosylation of the serines and threonines and addition of sulphate ions to the tyrosine residues. Following maturation, the protein is subsequently secreted into the plasma in the form of 2 chains, of 210 kDa (up to residue 1648) and 80 kDa (from residue 1649 to residue 2332), which are joined by a divalent ion, with the lighter chain being linked non-covalently to the von Willebrand factor (vWf) by its N-terminal end (1 molecule of vWf per molecule of FVIII). In the plasma, this complex is stabilized by hydrophobic and hydrophilic bonds in the presence of a 50-fold excess of vWf. This latter is reported to inhibit the attachment of FVIII to phospholipids. The fact that FVIII binds to the platelets has been established, although the presence of specific receptors expressed on the surface of the platelets has not been clearly demonstrated (Nesheim et al., 1993). Following its attachment to the membrane phospholipids, it is reported to unmask high-affinity binding sides for FIXa (Bardelle et al., 1993).
FVIII is made up of three structural domains, A, B and C (Kaufman R J, 1992; Bihoreau et al., 1992) which are arranged in the order A1:A2:B:A3:C1:C2 (FIG. 1). The A domains possess more than 40% homology and are also homologous to ceruloplasmin. 30% homology also exists between the A domains of factor V and FVIII. The C domain occurs twice and is reported to be able to bind glycocon-jugates and phospholipids having a net negative charge (Kemball-Cook and Barrowcliffe (1992); Fay, P J, 1993)). It exhibits homology with lectins which are able to bind to negatively charged phospholipids. The platelet attachment site has been located in this region (C2 domain) (Foster et al., (1990)). While domain B, which represents more than 40% of the mass of FVIII, does not have any known specific activity, it could play a subtle role in the regulation of FVIII by protecting it, for example, from the action of thrombin. It does not possess any known homology with other proteins.
It possesses 19 glycosylation sites out of the 25 which have been identified in FVIII. Comparison of the amino acid sequences of human and porcine FVIII reveals major differences within this B region. Nevertheless, porcine FVIII is used effectively for treating haemophilics exhibiting inhibitors. These observations have led to the construction of an FVIII gene from which the part encoding this B region has been deleted and which can be used to produce a deleted recombinant FVIII which is intended for the treatment of haemophilia.
Using immunopurification, different forms of active FVIII have been isolated which all possess a light chain of 80 kDa and whose heavy chain can have a molecular weight of between 210 and 90 kDa. These forms are reported to be derived by progressive degradation of the C-terminal end of the heavy chain. The binding of the two chains is non-covalent and results from a divalent metallic ion (Me++) bond between the responsible residues in domains A1 and A3. After formation of the activated complex (50-45 kDa) (heavy chain having accessible A2 domain) and 70 kDa (light chain), an inactivation phase is observed, probably as a consequence of prolonged contact with thrombin and dissociation of the 50 kDa and 45 kDa fragments. FVIIIa is also inactivated by activated protein C (APC) following proteolysis of the heavy chain. This inactivation is accelerated if the FVIIIa is attached to a phospholipid surface. This down-regulation of the activity of FVIIIa is reported to depend on a phosphorylation by a platelet enzyme (Kalafatis et al., (1990)).
Most of the epitopes which are recognized by the various murine monoclonals which have been isolated to date do not appear to be located in the “functional sites” of FVIII. Some epitopes have been identified which are recognized by antibodies which have an effect on the activity of FVIII (inhibition of the chromogenic and/or clotting tests).
These antigenic determinants consist of fragments 351-365 (A1 domain—heavy chain), 713-740 (A2 domain), 1670-1684 (A3 domain—light chain) (NH2 end of the light chain) or else 2303-2332 (C2 domain—light chain) (Foster C, (1990)), fragments 701-750 (Ware et al. (1989)), 1673-1689 (Leyte et al. (1989)), 330-472, 1694-1782 (EP-0 202 853), 322-740 and 2170-2322 (Scandella et al. (1992)).
The antibodies which recognize these various sites interfere, respectively, with the activation of FVIII, the binding of vWf or the binding of phospholipids.
Other antibodies, which do not inhibit standard activity tests in vitro, can exert an influence on the behaviour of FVIII with the other constituents of the coagulation cascade while attaching themselves to sites in the molecule which are at a substantial distance from the active sites. These antibodies, thus modified, can interfere with the natural state of folding of FVIII by altering some of its properties (“allosteric model”).
These mapping experiments make use of peptides which are synthesized by FVIII gene fragments which are cloned into E. coli and only provide an approximate idea of the location of the antigenic determinants which are recognized by these monoclonal antibodies. Thus, the sizes of the identified fragments range between 30 and 100 amino acids.
At present, it is necessary to crystallize a protein and analyse it with X rays in order to identify its antigenic sites unambiguously. Unfortunately, no data are available for FVIII, whose high molecular weight is a major handicap with regard to crystallization.
The antigenic regions coincide with the hydrophilic character of these regions: the more the oligopeptide sequence is exposed to the external medium (situated on the surface), the more this part is capable of being recognized in an immune reaction. By contrast, the hydrophobic parts, which are generally situated in the interior of the protein, are not considered to be very antigenic.
Currently, a predominant notion among haemophilic patients, clinicians and “fractionators” is that of having available a purified FVIII which is devoid of all pathogenic plasma contaminants and secondary effects.
However, whether after immunopurification using murine monoclonal antibodies or after obtaining it by genetic recombination in mammalian cells, highly purified FVIII is extremely unstable for reasons which are not apparent. In order to stabilize it, substantial quantities of human plasma albumin are added during the course of purification, such that the final specific activity is of the order of 2-3 U/mg of protein. The same applies to rFVIII which is coexpressed with the von Willebrand factor, which is a natural stabilizer, in CHO cells. These data appear to suggest that the purification steps exert an influence on the FVIII molecule, with these steps being able to interfere with its natural state of folding, to introduce confirmational changes which are more or less stable and to reveal new potential epitopes following infusion into the patient.
According to the authors (Ljung et al. (1992); Sultan et al., (1992); Lorenzo et al. (1992)), one of the serious complications which is seen in from 5 to 50% of the haemophilics who are given multiple therapeutic infusions of FVIII is the appearance of antibodies (inhibitors) which inactivate FVIII and render ineffective any subsequent injection of FVIII.
The spontaneous appearance of autoantibodies having a pathological anti-FVIII activity is rare in non-haemophilics (prevalence: 10−5) and has been reported in elderly individuals, in those exhibiting immunological disorders and in post-partum individuals (Kessler (1991), Hultin (1991)). A multi-centre study which was carried out on 3,435 haemophilic patients shows that all the age groups are affected, including patients of less than 5 years old. The majority (82%) display a very high response (>10 BU) (Sultan et al. (1992)). While these anti-FVIII antibodies have been reported to consist essentially of IgG antibodies of the IgG4 type, IgG2 (Gilles et al. (1993)b), IgA and IgM antibodies have also been described (Lottenburg et al. (1987)). They react weakly with purified heterologous FVIII molecules from other mammals (Bennett, B et al. (1972)). At the present time, it is not known what induces the appearance of the inhibitors in some haemophilics. If there is an association between the severity of the deletion of the gene and the development of an immune response which no longer recognizes FVIII as a self protein, this association is only demonstrated in a minority of patients. It has not been possible to demonstrate any specific host susceptibility which is linked to genetic markers, such as, for example, a preferential association with certain determinants of the MEC class II complex (Hoyer (1991)), without a doubt because not all the FVIII epitopes which are recognized by specific antibodies have yet been determined. It also appears that the different methods of preparing FVIII could exert an influence on its structure, its physicochemical properties or its natural microenvironment (Vermeylen, J and Peerlinck (1991); Gomperts, et al. (1992); Peerlinck et al. (1993)). Barrowcliffe et al. (1983) have demonstrated that phospholipids protect the procoagulatory activity from inactivation by specific human antibodies. The presence of natural anti-FVIII antibodies in 17% of healthy donors (screening carried out on 500 plasma donations) without any pathological symptoms demonstrates the importance of becoming better acquainted with the three-dimensional structural appearance assumed by physiological FVIII (Ciavarella and Schiavoni (1992)).
Transfusion which has been studied on mixed lymphocyte cultures, in animal models and during clinical trials has demonstrated modification of the immuno-modulation in the transfused subject, inducing an allo-immunization and also a down-regulation of some immune functions. It expresses itself in the form of suppressor cells, anti-idiotype antibodies or a decrease in NK cells. It is as if a certain degree of tolerance was being induced. These effects can be reversed by infusing interleukin 2 (IL-2) (Triulzi et al., 1990). In vitro, an inhibitory effect on the secretion of IL-2 as well as the proliferation of peripheral blood mononuclear cells are obtained in the presence of a cryoprecipitate or relatively impure preparations of FVIII (from 0.5 to 10 U/mg of protein) (Madhok et al., 1991; Wadhwa, M et al., 1992). These effects are not observed in the presence of rFVIII or FVIII which have been purified by immunoaffinity. This latter preparation is reported to have an activating effect on T cells (Madhok et al., 1991). However, it is not possible to extrapolate these findings directly to an in vivo situation.
No experimental model exists which makes it possible to forecast the immunogenicity or the immuno-modulatory effect of the FVIII preparations, or the susceptibility of the host, before they have been administered clinically. This model becomes an absolute necessity in the face of the increase in the frequency of the appearance of anti-FVIII antibodies in current clinical trials which make use of FVIII preparation which are of very high specific activity and which have been obtained either by immunopurification or by DNA manipulation techniques (Seremetis et al. (1991)). In addition, Aledorf (1993) has demonstrated that when these two types of preparation are used in naive subjects who have not previously been transfused (PUPS), an inhibitor prevalence is observed which amounts to up to 27%.